CN113563275A - 一种选择性靶向cdk9的氨基嘧啶类衍生物的制备及其应用 - Google Patents
一种选择性靶向cdk9的氨基嘧啶类衍生物的制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种选择性靶向CDK9的氨基嘧啶类衍生物的制备及其应用，本发明还公开了此类化合物的制备方法及其在预防和/或治疗肿瘤相关疾病中的应用，包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌等；该类化合物可以有效地选择性抑制CDK9蛋白的活性，同时在MV4‑11，MCF‑7和MOLM‑13等癌细胞中表现出显著的抗肿瘤活性。
Description
技术领域
本发明涉及到药物化学领域，具体包括可有效抑制CDK9蛋白活性的氨基嘧啶类衍生物及其制备方法和应用。
背景技术
细胞周期依赖性蛋白激酶(Cyclin-dependent kinases,CDKs)是细胞周期调控的关键的蛋白激酶，可有效调节DNA合成和有丝分裂。与相应的伴侣蛋白结合形成复合物是发挥CDK蛋白功能的必要条件。根据其主要功能，可将CDK分为周期性CDK(包括CDK1，CDK2，CDK3，CDK4和CDK6)和转录性CDK(包括CDK7，CDK8，CDK9，CDK11，CDK12，CDK13，CDK19等)。研究表明很多癌症存在“转录成瘾性”，因此，转录性CDK近来备受关注。其中，CDK9蛋白广泛参与了转录的起始、延伸和终止阶段，特别的是，CDK9可通过磷酸化RNA聚合酶II的CTD端进而有效地促使RNA转录延伸，是调节RNA转录的关键因子，可以有效调节下游蛋白水平，包括抗凋亡蛋白MCL-1和MYC。多项研究表明，CDK9蛋白的失调出现在多种肿瘤的发生发展中，包括各类白血病，前列腺癌、肺癌、肝癌、胃癌、乳腺癌等等。2018年，Cell期刊上发表文章指出，CDK9蛋白的抑制可激活被肿瘤抑制的基因，促进抑癌基因的表达和细胞分化。上述研究表明，寻找靶向抑制CDK9的小分子药物可能是一种有效的研发抗肿瘤药物的策略。
目前已有十余个CDK9抑制剂处于临床研究阶段，但其中大部分为非选择性的，进而导致很多难以预料的毒副作用。甚至有部分药物的临床试验因此被终止。由于CDK家族成员的高度同源性，想获得高选择性的CDK9抑制剂有一定困难。但鉴于目前的临床研究情况和进一步深入研究CDK9生物学作用的需求，选择性CDK9抑制剂的开发至关重要。也因此，近两年不断有选择性CDK9抑制剂进入临床研究，BAY-1143572是拜耳公司研发的第一个进入临床的选择性CDK9抑制剂。但由于嗜中性白血球减少症，BAY-1143572的临床试验已经被终止，导致这一副作用的原因暂没有相关报道。BAY-1251152是拜耳公司推出的第二代选择性CDK9抑制剂，目前该药已被转让给一家名Vincerx的制药公司，该化合物正处于临床研究中心。同时，在2017年左右，阿斯利康研发的AZD-4573也进入临床研究，这是有一个高选择性的CDK9抑制剂，在MV4-11小鼠异种移植模型中，有着极为显著的体内抗肿瘤活性。值得注意的是，近两年，国外BioTheryX公司的BTX-A51和Kronos Bio的KB-072，国内石药集团和千红药业均推出了自己的选择性CDK9抑制剂，由此可见，选择性CDK9抑制剂的开发已获得制药人的广泛关注。
发明内容
本发明的主要目的是寻找具有氨基嘧啶或氨基三嗪结构的，高选择性，良好的成药性质的CDK9小分子抑制剂。
本发明提供一种如通式I或式II所示的化合物或其药学上可接受的盐型：
其中，Ar选自如下基团：
X选自氢，氘，卤素，氰基，甲基或三氟甲基；
M和Y分别独立选自氮或碳；
L是价键或CH2；
R1选自如下基团：
本发明还提供一种如通式III所示的化合物或其药学上可接受的盐型，
其中：Ar，X，M，Y和R1如上所述。
本发明所述的药学上可接受的盐型是指通式I，II和III所示的化合物和药学上可接受的酸形成的盐，包括无机酸盐和有机酸盐。其中无机酸盐包括：盐酸、硫酸、磷酸、碳酸、碳酸氢根、硝酸、磷酸一氢根、磷酸二氢根，氢溴酸或氢碘酸；有机酸包括：马来酸、酒石酸、柠檬酸、甲磺酸、琥珀酸、乙酸、对甲苯磺酸、扁桃酸、异丁酸、丙二酸等。
在一些具体的实例中，本发明提供如下任一所述的化合物或其药学上可接受的盐：
本发明还公开本发明所述化合物的合成方法，选自以下任一：
本发明还提供一种药物组合物，包括本发明所述的化合物，或其药学上可接受的盐型或者前药。
本发明还提供本发明所述化合物或其药学上可接受的盐型在制备CDK9抑制剂药物中的应用，发明人发现本发明所述化合物或其药学上可接受的盐型可有效抑制CDK9蛋白活性或应用于以CDK9为靶点的小分子药物的发现。
本发明还提供本发明所述化合物或其药学上可接受的盐型在制备抗病毒药物或者抗肿瘤药物中的应用。
在本发明的一些实施例中，所述的病毒包括：HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒。
本发明的一些实施例中，所述的肿瘤包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌。
本发明所述的化合物为具有较好的CDK9抑制活性的苗头化合物。生物活性评价显示，所设计化合物具有显著的CDK9抑制活性，对CDK家族成员具有较好的选择性，同时对MV4-11，MCF7，MOLM13等肿瘤细胞具有良好的抗增殖活性。
具体实施方式
下面通过具体的实施例对本发明做进一步的详细说明，但并不将本发明限制在所举的实施例范围之中。
除非做出说明，实施列中的化合物可通过以下路线进行合成，具体如下：
路线1：
中间体3的合成：
将2,4-二氯三嗪(1,3.28g,0.024mol)溶于35ml四氢呋喃和异丙醇的混合溶剂中(1:1)，在氮气保护下降温至-40℃，然后缓慢滴加DIPEA(17.85mol,0.088mol)，再将对氨基苯甲酸(2,3g,0.022mol)溶解于35ml四氢呋喃和异丙醇的混合溶剂中(1:1)并缓慢滴加至反应液中，上述过程均控制温度在-40℃以下。滴加完毕后，保持该温度继续反映约2小时，待TLC检测原料2反应完全，停止反应，将反应液浓缩，使用乙酸乙酯与甲醇混合溶剂(30:1)重结晶，可得约2.5g淡黄色固体(3)，收率45％。HRMS[ESI]+,calcd for C10H7ClN4O2[M-H]-,249.0258,found 249.0250.
中间体4的合成：
将4-氟-2-甲氧基苯硼酸(2g,8mmol)，中间体3(1.63g,9.6mmol)溶解于20ml二氧六环中，然后加入Pd(PPh3)2Cl2(230mg,0.32mmol),碳酸钠水溶液(20ml,2N)，使用氮气置换空气三次后升温至100℃反应，约10小时后，TLC检测中间体3反应完全，停止反应，待反应液冷却后，使用稀盐酸调节PH至酸性，然后使用乙酸乙酯萃取三次，饱和氯化钠水溶液洗涤1次，无水硫酸钠干燥，抽滤，浓缩，柱层析分离后得1.69g白色固体(4)，收率53％。HRMS[ESI]+,calcd for C17H13FN4O3[M-H]-,339.0972,found 339.0961.
化合物X1的合成：
将中间体4(0.5mmol)，HATU(1.75mmol)溶解在4ml的四氢呋喃中，然后加入DIPEA(1mmol)，室温搅约15分钟，然后加入含裸露氨基的基团侧链(R1，0.5mmol)，室温搅拌反应1-3小时，待TLC检测中间体4反应完全，停止反应，柱层析分离纯化后可得化合物X1。
路线2：
中间体8的合成：
将2,4-二氯-5-氟嘧啶(6,11mmol)，中间体7(10mmol)溶解于20ml二氧六环中，然后加入Pd(PPh3)2Cl2(3mmol),碳酸钠水溶液(20ml,2N)，使用氮气置换空气三次后升温至100℃反应，约2小时后，TLC检测中间体6反应完全，停止反应，待反应液冷却后，加入适量水，然后使用乙酸乙酯萃取三次，饱和氯化钠水溶液洗涤1次，无水硫酸钠干燥，抽滤，浓缩，最后使用柱层析分离纯化得中间体8。
中间体9的合成：
将醋酸钯(0.05mmol)和XPhos(0.06mmol)溶解于6ml二氧六环中，氮气置换空气三次后，于室温搅拌约15分钟备用。将中间体8(1mmol)，对氨基苯甲酸(1mmol)，碳酸铯(3mmol)加入100ml三颈瓶中，然后计入上述新制的催化剂，氮气置换空气三次，然后升温至110℃回流反应，反应2-4小时后，TLC检测反应物消失，停止反应，冷却，使用稀盐酸调节pH至酸性，加入适量水，使用二氯甲烷萃取三次，饱和氯化钠洗涤1次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体9。
化合物X2的合成：
化合物X2的合成以中间体9和含裸露氨基的基团侧链为原料，合成方法同化合物X1的合成。
实施例1-17所述化合物的制备可以通过路线1的方法进行。除作特殊说明，其中间体合成步骤不再赘述。
实施例1：化合物LW-001
1H NMR(300MHz,CDCL3):δ(ppm)＝8.83(s,1H),8.00(s,1H),7.89-7.81(m,4H),7.65(s,1H),6.82-6.76(m,2H),3.96(s,3H),3.83(s,4H),3.65(d,J＝6.0Hz,2H),2.75(s,2H),2.67-2.50(m,4H).
实施例2：化合物LW-002
1H NMR(300MHz,CDCL3):δ(ppm)＝9.04(s,1H),8.67(s,1H),7.86-7.80(m,5H),7.65(s,1H),6.89-6.74(m,2H),3.98(s,3H),3.65(d,J＝6.0Hz,2H),3.01(s,4H),2.51-2.45(m,6H),2.14(s,3H),1.65-1.62(m,2H).
实施列3：化合物LW-003
1H NMR(300MHz,CDCL3):δ(ppm)＝8.83(s,1H),8.02(s,1H),7.88-7.81(m,4H),7.73(s,1H),7.12(s,1H),6.82-6.76(m,2H),3.96(s,3H),3.65-3.59(m,2H),2.76-2.65(m,10H),2.39(s,3H).
实施列4：化合物LW-004
1H NMR(300MHz,CDCL3):δ(ppm)＝8.91(s,1H),8.07(s,1H),7.87-7.80(m,4H),7.71(s,1H),7.15(s,1H),6.83-6.78(m,2H),3.96(s,3H),3.60-3.53(m,2H),2.34-2.30(m,3H),2.18(s,3H),2.02-1.96(m,6H).
实施列5：化合物LW-005
1H NMR(300MHz,CDCL3):δ(ppm)＝8.98(s,1H),8.03(s,1H),7.87-7.82(m,4H),7.73(s,1H),7.12(s,1H),6.83-6.77(m,2H),3.96(s,3H),3.60-3.54(m,2H),2.51-2.47(m,2H),2.42(s,4H),1.49-1.39(m,6H).
实施列6：化合物LW-006
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.6(s,1H),9.93(s,1H),8.88(s,1H),8.02--7.95(m,5H),7.21-7.12(m,4H),6.97-6.90(m,1H),6.50-6.46(m,1H),3.95(s,3H),2.90(s,6H).
实施列7：化合物LW-007
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.67(s,1H),9.00(s,1H),8.90(s,1H),8.76(s,1H),8.60(d,J＝6.0Hz,1H),8.14-7.95(m,7H),7.65-7.61(m,1H),7.18-7.14(d,J＝12.0Hz,1H),6.98-6.92(m,1H),3.96(s,3H).
实施列8：化合物LW-008
1H NMR(300MHz,DMSO-d6):δ(ppm)＝12.67(s,1H),10.53(s,1H),8.85(s,1H),8.24(d,J＝6.0Hz,2H),7.96(br,3H),7.52-7.44(m,2H),7.27-7.10(m,3H),6.96-6.89(m,1H),3.92(s,3H),3.71(s,1H).
实施列9：化合物LW-009
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.57(s,1H),8.89(s,1H),8.85(s,1H),8.35(s,1H),8.00(br,2H),7.88(d,J＝9.0Hz,2H),7.19(d,J＝9.0Hz,1H),6.99-6.93(m,1H),3.97(s,3H),2.63-2.54(m,8H),1.04(t,J＝6Hz,6H).
实施列10：化合物LW-010
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.64(s,1H),9.26(s,1H),8.91(s,1H),8.66(t,J＝6.0Hz,1H),8.02(br,2H),7.92(d,J＝9.0Hz,3H),7.21(d,J＝9.0Hz,1H),7.00-6.94(m,1H),3.97(s,3H),3.64(d,J＝6.0Hz,2H),3.42(s,2H),3.30(d,J＝6.0Hz,2H),2.89(s,6H).
实施例11：化合物LW-011
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.85(s,1H),8.68(s,1H),8.64(t,J＝6.0Hz,1H),7.98-7.88(m,5H),7.16(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.98(s,3H),3.72(m,2H)
实施例12：化合物LW-012
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.86(s,1H),8.68(s,1H),8.65(t,J＝6.0Hz,1H),7.99-7.89(m,5H),7.18(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99(s,3H),3.75(m,2H)
实施例13：化合物LW-013
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.85(s,1H),8.68(s,1H),8.65(t,J＝6.0Hz,1H),7.98-7.89(m,5H),7.17(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99,(m,1H)3.97(s,3H),3.52(d,J＝12Hz,2H),1.27(m,1H)
实施例14：化合物LW-014
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.84(s,1H),8.68(s,1H),8.69(t,J＝6.0Hz,1H),7.98-7.88(m,5H),7.13(d,J＝12.0Hz,1H),6.98-6.92(m,1H),3.98(s,3H),3.72(t,J＝6.0Hz,2H),3.28(s,3H),3.26(m,2H)
实施例15：化合物LW-015
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.87(s,1H),8.67(s,1H),8.66(t,J＝6.0Hz,1H),7.98-7.89(m,5H),7.18(d,J＝12.0Hz,1H),6.99-6.92(m,1H),3.94(s,3H),3.83(s,2H),3.52(s,3H)
实施例16：化合物LW-016
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.87(s,1H),8.69(s,1H),8.67(t,J＝6.0Hz,1H),7.99-7.87(m,5H),7.14(d,J＝12.0Hz,1H),6.99-6.92(s,1H),5.32(s,1H),4.94(s,1H),4.21(m,1H),3.59(t,J＝9.0Hz,2H),3.52(t,J＝9.0Hz,2H)
实施列17：化合物LW-017
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.89(s,1H),8.66(s,1H),8.64(t,J＝6.0Hz,1H),7.93-7.89(m,5H),7.19(d,J＝12.0Hz,1H),6.97-6.93(m,1H),3.97(s,3H),3.70-3.67(m,5H),1.99(t,J＝9.0Hz,2H),1.74(t,J＝9.0Hz,2H)
实施列18：化合物LW-018
化合物LW-018的合成：
(1)LW-018-1的合成：LW-018-1的合成可参考路线一的合成方法。
(2)将上述得到的LW-018-1(0.35mmol)溶解在2ml二氯甲烷中，然后加入三氟乙酸(5ml)，氮气保护，常温下反应约2小时，TLC检测反应完全，停止反应，使用2N的碳酸钠水溶液调节pH至碱性，并加入适量水和二氯甲烷搅拌约15分钟，分离有机层，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化，得约105mg固体(LW-018)，收率63％1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.86(s,1H),8.68(s,1H),8.65(t,J＝6.0Hz,1H),7.98-7.87(m,5H),7.15(d,J＝12.0Hz,1H),6.97-6.92(m,1H),3.99(s,3H),3.62(m,1H),2.89-2.78(m,4H),2.0(s,1H),1.89(m,2H),1.64(m,2H)
实施例19-20所述化合物的制备可参考实施例18中的路线和方法进行。
实施列19：化合物LW-019
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.89(s,1H),8.66(s,1H),8.64(t,J＝6.0Hz,1H),7.98-7.86(m,5H),7.18(d,J＝12.0Hz,1H),6.97-6.93(m,1H),3.98(s,3H),3.64(m,1H)，2.54(m,4H)1.44-1.73(m,7H)
实施列20：化合物LW-020
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.88(s,1H),8.70(s,1H),8.67(d,J＝6.0Hz,1H),8.50(s,1H),7.89-7.78(m,5H),7.36(d,J＝12Hz,1H),7.05-6.98(m,1H),3.90(s,3H),3.82(t,J＝7.8Hz,1H),2.83-2.69(m,4H),1.79-1.67(m,3H),1.67-1.57(m,5H),1.48-1.36(m,2H).
实施例21-22所述化合物的制备可参考路线一的合成方法。
实施列21：化合物LW-021
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.88(s,1H),8.78(s,1H),8.60(t,J＝5.9Hz,1H),8.02-7.89(m,5H),7.05(d,J＝11.5Hz,1H),6.95-6.83(m,1H),3.90(s,3H),3.85(m,1H),3.59(t,J＝8.5Hz,4H),2.55-2.42(m,5H),1.88–1.70(m,4H),1.69-1.54(m,4H).
实施列22：化合物LW-022
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.5(s,1H),8.90(s,1H),8.75(s,1H),8.63(t,J＝5.8Hz,1H),7.99-7.86(m,5H),7.10(d,J＝11.8Hz,1H),6.90-6.81(m,1H),3.99(s,3H),3.01-2.89(m,1H),2.75-2.61(m,1H),1.77-1.64(m,4H),1.61-1.45(m,4H).
实施列23：化合物LW-023
化合物LW-023的合成：
(1)化合物LW-023-1的合成可参考路线一的合成方法。
(2)将上述所得的LW-023-1(0.42mmol)溶解在5ml的甲醇中，然后加入50％的氢氧化钠水溶液(3ml)，室温搅拌2小时，TLC检测反应完全，停止反应，使用稀盐酸调节pH至酸性，析出白色固体，抽滤后，滤饼干燥，使用柱层析分离得化合物LW-017。1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.35(s,1H),8.85(s,1H),8.70(s,1H),8.66(t,J＝6.0Hz,1H),7.98-7.89(m,5H),7.15(d,J＝12.0Hz,1H),6.98-6.92(m,1H),3.9-3.82(m,4H),2.50-2.41(m,1H),1.91-1.68(m,6H),1.65-1.54(m,2H).
实施例24-29所述化合物的制备可参考路线1的合成方法，只需将路线一种的对氨基苯甲酸(中间体2)替换成对氨基苯乙酸(实施例24)或3-氨基苯甲酸(实施例25)或3-氨基苯乙酸(实施例26-29)即可。
实施列24：化合物LW-024
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.86(s,1H),8.71(s,1H),8.67(t,J＝6.0Hz,1H),7.95-7.82(m,5H),7.10(d,J＝12.0Hz,1H),6.87-6.78(m,1H),3.91(s,3H),3.56-3.43(m,5H),3.40(s,2H),2.01-1.91(m,2H),1.88-1.76(m,2H).
实施列25：化合物LW-025
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.85(s,1H),8.71(s,1H),8.11(d,J＝2.4Hz,1H),8.05(s,1H),7.65-7.48(m,2H),6.89(t,J＝6.0Hz,1H),6.75-6.64(m,3H),3.89(s,3H),3.55(d,J＝6.0Hz,2H),3.28(d,J＝5.7Hz,2H),2.84(s,6H).
实施列26：化合物LW-026
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.0(s,1H),8.86(s,1H),8.23(d,J＝2.5Hz,1H),8.02(s,1H),7.63-7.46(m,2H),6.89(t,J＝5.0Hz,1H),6.85-6.75(m,3H),3.91(s,3H),3.64(d,J＝6.0Hz,2H),3.52(s,2H),3.29(d,J＝6.0Hz,2H),2.86(s,6H).
实施列27：化合物LW-027
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.97(s,1H)8.82(s,1H),8.11(d,J＝2.4Hz,1H),7.96(s,1H),7.52-7.37(m,2H),6.88(t,J＝5.0Hz,1H),6.83-6.70(m,3H),3.92(s,3H),3.80(s,2H),3.72(t,J＝4.6Hz,4H),3.59(t,J＝5.6Hz,2H),2.61(t,J＝6.0Hz,2H),2.51(t,J＝4.6Hz,4H).
实施列28：化合物LW-028
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.01(s,1H),8.81(s,1H),8.11(d,J＝2.7Hz,1H),7.90(s,1H),7.54-7.38(m,2H),6.89(t,J＝6.0Hz,1H),6.86-6.77(m,3H),3.88(s,3H),3.60(s,2H),3.45(t,J＝6.0Hz,4H),2.82(t,J＝6.7Hz,4H).
实施列29：化合物LW-029
1H NMR(300MHz,DMSO-d6):δ(ppm)＝8.90(s,1H),8.82(s,1H),8.07(d,J＝2.5Hz,1H),7.97(s,1H),7.53-7.37(m,2H),6.90(t,J＝5.7Hz,1H),6.85-6.79(m,3H),3.90(s,3H),3.65-3.57(m,5H),3.53(s,2H),1.87-1.72(m,2H),1.55-1.42(m,2H).
实施例30所述化合物的制备可参考路线一的合成方法，只需将路线一种的4-氟-2甲氧基苯硼酸替换成5-氟-2-甲氧基苯硼酸即可。
实施列30：化合物LW-030
实施例31-40的合成可参考路线2的合成方法。
实施列31：化合物LW-031
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.41(s,1H),8.81(s,1H),8.51(s,1H),7.65-7.78(m,5H),7.35(s,1H),6.99(t,J＝6.0Hz,1H),3.80(s,3H),3.62(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.20(s,6H).
实施列32：化合物LW-032
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.38(s,1H),8.86(s,1H),7.90(s,1H),7.62-7.80(m,5H),7.35(d,J＝6.0Hz,1H),6.99(t,J＝12.0Hz,1H),3.76(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.20(s,6H).
实施列33：化合物LW-033
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.01(s,1H),8.51(s,1H),8.21(s,1H),7.62-7.58(m,5H),7.31(d,J＝6.0Hz,1H),6.97(t,J＝12.0Hz,1H),3.83(s,3H),3.61(t,J＝6.0Hz,2H),2.46(t,J＝6.0Hz,2H),2.38(s,3H),2.22(s,6H).
实施列34：化合物LW-034
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.44(s,1H),8.86(s,1H),8.83(s,1H),7.78-7.60(m,5H),7.33(d,J＝6.0Hz,1H),6.96(t,J＝12.0Hz,1H),3.80(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.22(s,6H).
实施列35：化合物LW-035
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.40(s,1H),8.81(s,1H),8.53(s,1H),7.71-7.65(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.80(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.22(s,6H).
实施列36：化合物LW-036
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.46(s,1H),8.53-8.48(m,2H),7.62-7.70(m,4H),7.52(s,1H),7.32(t,J＝6.0Hz,1H),7.11(t,J＝6.0Hz,1H),3.82(s,3H),3.50(t,J＝12.0Hz,2H).
实施列37：化合物LW-037
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.40(s,1H),8.86(s,1H),8.53(s,1H),7.71-7.62(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.83(s,3H),3.63-3.51(m,6H),2.52-2.41(m,6H).
实施列38：化合物LW-038
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.42(s,1H),8.81(s,1H),8.40(s,1H),7.73-7.66(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.80(s,3H),3.63(t,J＝6.0Hz,2H),2.47(t,J＝6.0Hz,2H),2.40(s,3H),2.18(s,6H).
实施列39：化合物LW-039
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.37(s,1H),8.81(s,1H),8.40(s,1H),7.69-7.65(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.83(s,3H),3.62(t,J＝6.0Hz,2H),3.50(t,J＝6.0Hz,4H)2.52(t,J＝6.0Hz,2H),2.43-2.40(m,7H).
实施列40：化合物LW-040
1H NMR(300MHz,DMSO-d6):δ(ppm)＝9.40(s,1H),8.40(s,1H),7.80(s,1H),7.72-7.62(m,4H),7.52(s,1H),7.30(d,J＝6.0Hz,1H),7.11(d,J＝6.0Hz,1H),3.83(s,3H),3.57-3.70(m,5H),2.40(s,3H),1.97-1.74(m,4H).
实施例41所述化合物的合成可以参考路线一的合成方法。
实施列41：化合物LW-041
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.67(s,1H),9.26(s,1H),8.93(s,1H),7.91(d,J＝9.0Hz,2H),7.68(d,J＝6.0Hz,1H),7.47(t,J＝9.0Hz,1H),7.40(s,1H),7.23(s,2H),3.92(s,3H),3.63(d,J＝6.0Hz,2H),3.30(d,J＝6.0Hz,2H),2.88(s,6H).
实施例42-44所述化合物的合成可参考路线2的合成方法。
实施列42：化合物LW-042
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.51(s,1H),9.16(s,1H),8.53(s,1H),7.81(d,J＝9.0Hz,2H),7.63(d,J＝6.0Hz,1H),7.50(t,J＝9.0Hz,1H),7.41(s,1H),7.25(d,J＝9.0Hz,2H),3.95(s,3H),3.65(d,J＝6.0Hz,2H),3.23(d,J＝6.0Hz,2H),2.89(s,6H).
实施列43：化合物LW-043
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.50(s,1H),9.11(s,1H),8.54(s,1H),7.88(d,J＝9.0Hz,2H),7.62(d,J＝6.0Hz,1H),7.51(t,J＝9.0Hz,1H),7.38(s,1H),7.15(d,J＝9.0Hz,2H),3.76(t,J＝6.0Hz,4H),3.70-3.59(m,2H),2.64(t,J＝6.0Hz,2H),2.54(s,4H).
实施列44：化合物LW-044
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.50(s,1H),9.11(s,1H),8.54(s,1H),7.88(d,J＝9.0Hz,2H),7.62(d,J＝6.0Hz,1H),7.51(t,J＝9.0Hz,1H),7.38(s,1H),7.15(d,J＝9.0Hz,2H),4.25(q,J＝3.0Hz,1H),4.01(dt,J1＝12.0Hz,J2＝3.0Hz,2H),3.61(t,J＝9.0Hz,2H),2.11-1.96(m,4H).
实施列45：化合物LW-045
化合物LW-045的合成路线如下：
中间体11的合成：
将2,4-二氯-5-氟嘧啶(6,11mmol)，中间体10(10mmol)溶解于20ml的DMF中，降温至0℃以下，然后分三次加入钠氢(15mmol)，加完后移至室温搅拌反应约3小时，TLC检测中间体10反应完全，停止反应，使用适量冰水淬灭反应，然后使用乙酸乙酯萃取3次，饱和氯化钠水溶液洗涤2次，无水硫酸钠干燥，抽滤，浓缩，最后使用柱层析分离纯化得中间体11。HRMS[ESI]+,calcd for C11H6ClFN4[M+H]+,249.0265,found 249.0251.
中间体12的合成可参考路线2中提供的制备方法。
化合物LW-046的合成以中间体12原料，具体合成方法可参考路线2提供的制备方法，收率45％。1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.68(s,1H),8.98(s,1H),8.67(d,J＝6.0Hz,1H),8.21(s,1H),8.09(s,1H),7.88(d,J＝9.0Hz,2H),7.62-7.59(m,2H),7.51(t,J＝9.0Hz,1H),7.25(t,J＝9.0Hz,2H),3.79(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J＝6.0Hz,2H),2.54(s,4H).
实施列46：化合物LW-046
化合物LW-046的合成路线如下：
中间体13的合成可参考Waleed Minzel等人(doi:10.1016/j.cell.2018.07.045)提供的合成方法进行制备。LW-046的合成以中间体6和13为起始原料，合成方法参考路线提供的路线即可。1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.58(s,1H),8.98(s,1H),8.09(s,2H),7.88(d,J＝9.0Hz,2H),7.62-7.59(m,2H),3.81(s,3H),3.70(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J＝6.0Hz,2H),2.54-2.51(m,6H),0.81-0.77(m,1H),0.55-0.50(m,4H).
实施例47-49所述化合物的合成可参考实施例46所述的合成方法与路线。
实施列47：化合物LW-047
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.57(s,1H),8.88(s,1H),8.05(s,2H),7.88(d,J＝9.0Hz,2H),7.63-7.59(m,2H),4.25(q,J＝3.0Hz,1H),4.01(dt,J1＝12.0Hz,J2＝3.0Hz,2H),3.80(s,3H),3.61(t,J＝9.0Hz,2H),2.55(d,J＝6.0Hz,2H),2.11-1.96(m,4H),0.80-0.75(m,1H),0.55-0.50(m,4H).
实施列48：化合物LW-048
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.56(s,1H),8.81(s,1H),8.03(s,2H),7.89(d,J＝9.0Hz,2H),7.63-7.57(m,2H),3.81(s,3H),3.72-3.69(m,4H),3.64(t,J＝6.0Hz,2H),2.64(t,J＝6.0Hz,2H),2.54-2.51(m,6H),2.15(s,3H),0.80-0.75(m,1H),0.55-0.50(m,4H).
实施列49：化合物LW-049
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.57(s,1H),8.91(s,1H),8.09(s,1H),7.99(s,1H),7.90(s,1H),7.80(d,J＝9.0Hz,2H),7.63-7.58(m,2H),3.80(s,3H),3.72(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.64(t,J＝6.0Hz,2H),2.54(t,J＝6.0Hz,4H).
实施例50-53所述化合物的制备可参考路线二的合成方法，只需要路线二中的对氨基苯甲酸替换成6-氨基烟酸即可。
实施列50：化合物LW-050
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.47(s,1H),8.85(d,J＝6.0Hz,1H),8.67(s,1H),8.12(s,1H),7.98(t,J＝9.0Hz,1H),7.78(t,J＝9.0Hz,1H),7.25(d,J＝12.0Hz,1H),6.98-6.92(m,2H),3.95(s,3H),3.65(d,J＝6.0Hz,2H),3.30(d,J＝6.0Hz,2H),2.88(s,6H).
实施列51：化合物LW-051
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.51(s,1H),9.16(s,1H),8.53(s,1H),8.12(s,1H),7.98(t,J＝9.0Hz,1H),7.78(t,J＝9.0Hz,1H),7.26(d,J＝12.0Hz,1H),6.95-6.91(m,2H),3.91(s,3H),3.61(d,J＝6.0Hz,2H),3.33(d,J＝6.0Hz,2H),2.89(s,6H).
实施列52：化合物LW-052
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.53(s,1H),9.10(s,1H),8.55(s,1H),8.10(s,1H),7.99(t,J＝9.0Hz,1H),7.77(t,J＝9.0Hz,1H),7.36(d,J＝12.0Hz,1H),6.99-6.92(m,2H),3.91(s,3H),3.39(d,J＝6.0Hz,2H).
实施列53：化合物LW-053
1H NMR(300MHz,DMSO-d6):δ(ppm)＝10.57(s,1H),8.79(d,J＝6.0Hz,1H),8.66(s,1H),8.10(s,1H),7.99(t,J＝9.0Hz,1H),7.88(t,J＝9.0Hz,1H),7.21(d,J＝12.0Hz,1H),6.97-6.92(m,2H),3.95(s,3H),3.72(t,J＝6.0Hz,4H),3.69-3.61(m,2H),2.71-2.66(m,2H),2.54(t,J＝6.0Hz,4H).
以下为此发明部分化合物的生物活性评价，主要包括细胞水平与酶水平两个方面。
一、本发明部分化合物对多种癌细胞的增殖抑制活性
实验方法如下：
以MTT法测定化合物对CDK9高表达的肿瘤细胞(MV4-11，MCF7以及MOLM13)的细胞毒性：细胞以2000个/孔接种于96孔板中，待细胞贴壁后，吸除培养基，加入200uL稀释好的药物，37度，5％CO2培养72h，之后加入10uL/孔的MTT。37度孵育4个小时，吸去上清，每孔加入150uL的DMSO，多功能酶标仪492nm处检测吸光值。用GraphPad计算IC50并绘制细胞生长曲线。
二、本发明部分化合物对CDK9和CDK2蛋白的抑制活性
实验方法如下：
首先将化合物用100％DMSO配制成10mM储存液于低温下避光保存。激酶反应过程如下：
1)配制1×Kinase buffer。
2)化合物浓度梯度的配制：受试化合物测试浓度为5nM,20nM或100nM，复孔检测。在384孔板中配置成100倍终浓度的化合物。然后用Echo550转移250nl到384反应板中备用。阴性对照孔和阳性对照孔中分别加250nl的100％DMSO。
3)用1×Kinase buffer配制2.5倍终浓度的激酶溶液。
4)在化合物孔和阳性对照孔分别加10μl的2.5倍终浓度的激酶溶液；在阴性对照孔中加10μl的1×Kinase buffer。
5)1000rpm离心30秒，振荡混匀后室温孵育10分钟。
6)用1×Kinase buffer配制25/15倍终浓度的ATP和Kinase substrate的混合溶液。
7)加入15μl的25/15倍终浓度的ATP和底物的混合溶液，起始反应。
8)将384孔板1000rpm离心30秒，振荡混匀后室温孵育相应的时间。
9)加入30μl终止检测液停止激酶反应，1000rpm离心30秒，振荡混匀。
10)用Caliper EZ Reader读取转化率。
最后进行数据计算和分析：
1)抑制率计算公式：
其中：Conversion％_sample是样品的转化率读数；Conversion％_min：阴性对照孔均值，代表没有酶活孔的转化率读数；Conversion％_max：阳性对照孔均值，代表没有化合物抑制孔的转化率读数。
2)拟合量效曲线
以浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 5拟合量效曲线，从而得出各个化合物对酶活性的IC50值。
公式如下：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
本发明中部分化合物对CDK9和CDK2的抑制活性和其对MV4-11，MCF-7以及MOLM13细胞的抗增殖活性如下表1所示：
表1.本发明中部分化合物的酶活性和细胞活性
a：抑制率一栏中，括号内表示化合物浓度，单位为nM，如90(20)表示化合物在20nM浓度时对酶的抑制率为90％。
BAY-1143572的结构式如下所示：
根据表1所示数据，本发明所述的化合物对CDK9显示出有效的抑制活性，大部分化合物在20nM浓度下对CDK9的抑制率在70％以上，同时，与第一个进入临床的选择性CDK9抑制剂BAY-1143572相比，本发明所述化合物显示更优的CDK9抑制活性与细胞活性，比如化合物LW-010。相比于BAY1143572，LW-010的CDK9抑制活性获得一定改善的同时，其对MV4-11和MOML13细胞毒性显著提升(IC50＝20-40nM)。选择性上，化合物LW-008和LW-030在保持CDK9活性的同时，也显示出比阳性药更优或相似的选择性。其中化合物LW-008对CDK9和CDK2的IC50值分别为5.8nM和>5000nM,选择性超过800倍，显著优于BAY1143572(IC50:CDK9＝8.2nM，CDK2>1000nM,约121倍)。
Claims (10)
1.一种如通式I或式II所示的化合物或其药学上可接受的盐型：
其中，Ar选自如下基团：
X选自氢，氘，卤素，氰基，甲基或三氟甲基；
M和Y分别独立选自氮或碳；
L是价键或CH2；
R1选自如下基团：
2.一种如式III所示的化合物或其药学上可接受的盐型，：
其中Ar，X，M，Y和R1同权利要求1中所述。
3.如下任一所述的化合物或其药学上可接受的盐：
4.根据权利要求1～3任一项所述的化合物或其药学上可接受的盐型，其特征在于药学上可接受的盐型是指通式I，II或III所示的化合物和药学上可接受的酸形成的盐，包括无机酸盐或有机酸盐；优选的无机酸盐包括：盐酸、硫酸、磷酸、碳酸、碳酸氢根、硝酸、磷酸一氢根、磷酸二氢根，氢溴酸或氢碘酸；优选的，有机酸包括：马来酸、酒石酸、柠檬酸、甲磺酸、琥珀酸、乙酸、对甲苯磺酸、扁桃酸、异丁酸或丙二酸。
5.一种制备权利要求1所述化合物的制备方法，，选自以下任一：
6.一种药物组合物，其特征在于，包括权利要求1-4任一项所述的化合物，或其药学上可接受的盐型，及药学上可接受的载体。
7.权利要求1-4任一项所述的化合物，或其药学上可接受的盐型或权利要求6所述的组合物在制备CDK9抑制剂药物中的应用。
8.权利要求1-4任一项所述的化合物，或其药学上可接受的盐型或权利要求6所述的组合物在制备抗病毒药物或者抗肿瘤药物中的应用。
9.根据权利要求8所述的应用，其特征在于：所述的病毒包括：HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒。
10.根据权利要求8所述的应用，其特征在于：所述的肿瘤包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌。
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